The proposal will employ molecular cloning methods to characterize prostaglandin receptor subtypes in human erythroleukemia (HEL) cells and vascular smooth muscle cells and correlate the subtypes with patterns of cyclic AMP metabolism in both cell types. Prostaglandins are potent stimulators of cyclic AMP formation in platelets and vascular smooth muscle cells, leading to inhibition of platelet aggregation and relaxation of vascular smooth muscle so that therapeutic use of prostaglandins as antiplatelet agents may be offset by unwanted vasodilation. Platelets and smooth muscle cells may possess different prostaglandin stimulatory receptors that may be targeted selectively by different drugs. Platelet adenylate cyclase appears to be controlled by both stimulatory and Inhibitory prostaglandin receptors that act together to maintain platelet homeostasis while allowing the cells to respond rapidly to appropriate activators. Similar two-receptor control of cAMP formation occurs in HEL cells, a megaryocytic cell line with many properties of platelets. Prostaglandin receptors coupled to inhibition (EP3 subtype) and activation (EP2 subtype) of adenylate cyclase have been successfully cloned and expressed in this laboratory from HEL cells. Low stringency hybridization will be used to clone the stimulatory IP prostaglandin receptor. Cloned receptors will be used to probe receptor expression in HEL cells and in reconstitution studies to determine the validity of the two receptor model of prostaglandin regulation: stimulatory and inhibitory receptors will be transfected into CHO cells alone and together to examine corresponding patterns of cAMP formation. The activity of splice variants of the EP3 receptor subtype will be examined. Prostaglandin subtypes on vascular smooth muscle cells will be compared with HEL cell receptors by cloning techniques including Northern blot analysis, high fidelity PCR screening of vascular smooth muscle total RNA, and if necessary by cloning the corresponding vascular smooth muscle receptors. Further studies will correlate radioligand prostaglandin binding with receptor subtypes expressed on the cells and with patterns of cyclic AMP metabolism that can be analyzed by computer modeling.